重組全長副肌球蛋白在小鼠與水牛中杭日本血吸蟲感染的保護力研究
發(fā)布時間：2019-06-22 15:59
【摘要】：日本血吸蟲病是一種嚴重危害我國人民身體健康的寄生蟲病。根據2006年的調查資料,我國現有血吸蟲病人51萬多,有6000多萬人口受到血吸蟲病的威脅。近年,我國血吸蟲病防治以人畜同步化療為主,消滅易感地帶釘螺和加強健康教育為輔的綜合防治措施。多年的防治實踐證明,這一綜合措施雖然有效,但效果難以鞏固,不僅受到生態(tài)環(huán)境和防治力度變化的挑戰(zhàn),而且面臨著人群化療后再感染的困擾。單純依賴化療難以達到防治目標,鑒于疫苗在許多傳染病控制中無可比擬的作用,研發(fā)血吸蟲病疫苗,獲取化療短期效應與疫苗接種誘導的長期免疫預防作用相結合的雙重效果,將是未來有效防治血吸蟲病的主要奮斗目標。在我國,耕牛作為主要的保蟲宿主和傳染源在血吸蟲病的流行過程中起著重要的傳播作用。發(fā)展適合耕牛等主要保蟲宿主的動物疫苗對血吸蟲病的控制具有重要意義,此舉不僅可通過控制傳播降低人群血吸蟲病流行率,而且為人用血吸蟲病疫苗的研制提供一定的理論依據。 血吸蟲病疫苗的研究歷時70余年,大體上經歷了死疫苗、減毒活疫苗、基因工程疫苗、核酸疫苗研究階段。作為血吸蟲病疫苗候選分子之一的副肌球蛋白,在曼氏血吸蟲或日本血吸蟲的動物模型中均獲得了不錯的保護效果。相比其他血吸蟲病疫苗候選分子,副肌球蛋白誘生的保護力較為突出,蟲源性或重組副肌球蛋白分子片段,以及全長的DNA疫苗在小鼠、水牛等動物實驗中的減蟲率可以達到60%以上。然而,許多保護力實驗中所用的副肌球蛋白為成蟲的生化純化產物,或大腸桿菌中的重組表達產物。生化純化需要大量的蟲源,而重組表達一直未能解決全長片段純化困難、得率偏低的問題,長期以來,成為阻止其作為潛在疫苗快速推進到大規(guī)模研究,尤其是應用于大動物實驗的障礙。 免疫佐劑在調節(jié)免疫和提高疫苗效價方面具有重要作用,是疫苗獲得理想效果的一條重要途徑。良好的免疫佐劑不僅應該能夠加強抗原的免疫原性和免疫保護效果,同時也應該不引起過敏反應或激發(fā)自身免疫反應等副作用。本研究使用的重組副肌球蛋白分子表達于大腸桿菌,采用改進的純化方法后純度能夠達到95%以上,適于大規(guī)模制備與研究用途,重組蛋白結構和生物學特性與蟲源性副肌球蛋白一致;先后使用多種佐劑與重組副肌球蛋白聯合免疫小鼠,尋求最佳的疫苗組合;探索了ISA206佐劑與重組副肌球蛋白聯合免疫水牛后對日本血吸蟲感染的抵抗能力,以此來尋求適合于家畜(主要為耕牛)等保蟲宿主的副肌球蛋白疫苗組合。 本研究獲得如下結果: 1.大規(guī)模重組表達的全長副肌球蛋白分子的純度達到95%以上,其中LPS含量為0.07EU/ml,與蟲源性副肌球蛋白分子具有相同的二級結構和生物學功能,后者包括與免疫球蛋白的結合特性以及與膠原物質的結合特性。 2.在昆明鼠與C57BL/6小鼠模型中,rSj97-Alhydrogel未能誘導理想的保護力。 3.在C57BL/6小鼠模型中,就減蟲率與減卵率而言,與rSj97-FA組和rSj97-CpG-ODN組相比,rSj97-ISA206組對C57BL/6小鼠具有顯著的保護效果,分別為45.3%(P0.01)與35.4%(P0.05),該結果提示,rSj97-ISA206具良好的抗日本血吸蟲感染的保護效果。 4.在水牛模型中,rSj97-ISA206組相比ISA206佐劑組,所獲減蟲率為28.01%(P=0.443)、糞便蟲卵減少率為28.03%(P=0.584);相比感染對照組,減蟲率為16.08%(P=0.742)、肝臟減卵率為3.64%(P=0.963)、糞便減卵率為38.70%(P=0.462)。 5. rSj97在C57BL/6小鼠體內誘生出高水平特異性IgG2a與IgG1抗體,提示rSj97能誘導特異性Th1和Th2特征抗體亞類的產生,可能與誘導C57BL/6小鼠的保護力存在一定相關性。 6. rSj97免疫水牛后能誘生出高水平特異性IgG2與IgG1抗體,并誘生出Th1型優(yōu)勢應答,同時引起的體液免疫與細胞免疫應答可能是水牛產生保護力的因素之一。 本研究進一步豐富了副肌球蛋白的保護效果研究,尤其是在大動物體內的研究,為構建適合于水牛的副肌球蛋白疫苗組合提供了重要的基礎資料。
[Abstract]:Schistosomiasis japonica is a parasitic disease that is seriously harmful to the health of our people. According to the survey data of 2006, more than 550,000 of the present schistosomiasis has been made in China, and more than 60 million people have been threatened by the schistosomiasis. In recent years, the prevention and control of schistosomiasis in China is the main control measures for the prevention and control of the synchronous chemotherapy of the livestock and the livestock and the elimination of the Oncomelania hupensis and the strengthening of the health education. The years of prevention and control have proved that this comprehensive measure, though effective, is difficult to consolidate, not only is the challenge of the change of the ecological environment and the prevention and control, but also the problem of re-infection after the chemotherapy of the population. It is difficult to achieve the goal of prevention and cure by relying on chemotherapy alone, and in view of the incomparable role of the vaccine in the control of many infectious diseases, the double effects of the combination of the short-term effect of chemotherapy and the long-term immune prevention induced by the vaccine inoculation are obtained, It will be the main goal of the future effective control of the schistosomiasis. In our country, the cultivation of cattle as the main insect-protecting host and the source of infection plays an important role in the epidemic of schistosomiasis. The development of the animal vaccine suitable for the host of the main insect-protecting host such as the cultivated cattle is of great significance to the control of the schistosomiasis, which not only can reduce the prevalence rate of the schistosomiasis in the population by controlling the transmission, but also provides a certain theoretical basis for the development of the schistosomiasis vaccine for human use. The study of schistosomiasis vaccine has lasted for more than 70 years, and has generally undergone the study of dead vaccine, attenuated live vaccine, genetic engineering vaccine and nucleic acid vaccine. para. as the paramyosin of one of the candidate molecules of the schistosomiasis vaccine, a good protective effect is obtained in the animal models of the Schistosoma mansoni or the Schistosoma japonicum Compared with other schistosomiasis vaccine candidate molecules, the protection force of the paramyosin induced by the paramyosin is more prominent, the insect-derived or recombinant paramyosin molecule fragment, and the full-length DNA vaccine can reach 60% in animal experiments such as mice and buffalo. However, the secondary myosin used in many of the protection experiments is the biochemical purification product of the adult, or the recombinant expression in E. coli The biochemical purification requires a large number of entomogenous sources, and the recombinant expression has been unable to solve the problems of difficult purification and low yield of the full-length fragment, and has long been a barrier to the rapid advance of the full-length fragment to a large-scale study, in particular to a large animal experiment The immune adjuvant plays an important role in regulating immunity and improving the titer of the vaccine, and is a weight of the ideal effect of the vaccine. The good immune adjuvant not only can enhance the immunogenicity and the immune protective effect of the antigen, but also does not cause an allergic reaction or stimulate the self-immune reaction, and the like. The recombinant paramyosin molecule used in the study is expressed in E. coli, and the purity can reach more than 95% after the improved purification method, and is suitable for large-scale preparation and research purposes, the structure and biological characteristics of the recombinant protein and the worm-derived submuscle ball A combination of adjuvant and recombinant paramyosin was used to immunize mice, to find the best combination of the vaccine, and to explore the effect of the adjuvant of ISA206 and the recombinant paramyosin on the infection of Schistosoma japonicum. The ability to search for paramyosin phytophthora, which is suitable for the host of insect-protecting, such as cattle, and the like The combination of the seedlings. The present study was The results are as follows:1. The purity of the full-length paramyosin molecule expressed by the large-scale recombinant expression is over 95%, wherein the content of the LPS is 0.07 EU/ ml and the same secondary junction with the insect-derived paramyosin molecule Construction and biological functions, which include binding properties to immunoglobulins and with collagen the binding properties of the substance.2. In the Kunming mice and the C57BL/6 mouse model, rSj97-Hydrogel did not 3. Compared with rSj97-FA group and rSj97-CpG-ODN group, the rSj97-ISA206 group had significant protective effect on C57BL/6 mice compared with rSj97-FA group and rSj97-CpG-ODN group in the C57BL/6 mouse model. 4. Compared with the control group, the deinsectization rate was 16.08% (P = 0.742), the egg reduction rate of the liver was 3.64% (P = 0.963), and the egg reduction rate was 38. ..70% (P = 0.462). rSj97 induced a high level of specific IgG2a and IgG1 antibody in C57BL/6 mice, suggesting that rSj97 can induce the generation of specific Th1 and Th2-specific antibody subclasses, possibly related to the induction of C57BL/6 mice. 6. A high level of specific IgG2 and IgG1 antibodies can be induced by the rSj97 immune buffalo, and a Th1-type dominant response is induced, and humoral immunity and cellular immunity are induced. The response may be one of the factors that the buffalo produces a protective force. The study further enriched the study of the protective effect of paramyosin, especially in large animals, in order to build a suitable water buffalo
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392
本文編號：2504738
[Abstract]:Schistosomiasis japonica is a parasitic disease that is seriously harmful to the health of our people. According to the survey data of 2006, more than 550,000 of the present schistosomiasis has been made in China, and more than 60 million people have been threatened by the schistosomiasis. In recent years, the prevention and control of schistosomiasis in China is the main control measures for the prevention and control of the synchronous chemotherapy of the livestock and the livestock and the elimination of the Oncomelania hupensis and the strengthening of the health education. The years of prevention and control have proved that this comprehensive measure, though effective, is difficult to consolidate, not only is the challenge of the change of the ecological environment and the prevention and control, but also the problem of re-infection after the chemotherapy of the population. It is difficult to achieve the goal of prevention and cure by relying on chemotherapy alone, and in view of the incomparable role of the vaccine in the control of many infectious diseases, the double effects of the combination of the short-term effect of chemotherapy and the long-term immune prevention induced by the vaccine inoculation are obtained, It will be the main goal of the future effective control of the schistosomiasis. In our country, the cultivation of cattle as the main insect-protecting host and the source of infection plays an important role in the epidemic of schistosomiasis. The development of the animal vaccine suitable for the host of the main insect-protecting host such as the cultivated cattle is of great significance to the control of the schistosomiasis, which not only can reduce the prevalence rate of the schistosomiasis in the population by controlling the transmission, but also provides a certain theoretical basis for the development of the schistosomiasis vaccine for human use. The study of schistosomiasis vaccine has lasted for more than 70 years, and has generally undergone the study of dead vaccine, attenuated live vaccine, genetic engineering vaccine and nucleic acid vaccine. para. as the paramyosin of one of the candidate molecules of the schistosomiasis vaccine, a good protective effect is obtained in the animal models of the Schistosoma mansoni or the Schistosoma japonicum Compared with other schistosomiasis vaccine candidate molecules, the protection force of the paramyosin induced by the paramyosin is more prominent, the insect-derived or recombinant paramyosin molecule fragment, and the full-length DNA vaccine can reach 60% in animal experiments such as mice and buffalo. However, the secondary myosin used in many of the protection experiments is the biochemical purification product of the adult, or the recombinant expression in E. coli The biochemical purification requires a large number of entomogenous sources, and the recombinant expression has been unable to solve the problems of difficult purification and low yield of the full-length fragment, and has long been a barrier to the rapid advance of the full-length fragment to a large-scale study, in particular to a large animal experiment The immune adjuvant plays an important role in regulating immunity and improving the titer of the vaccine, and is a weight of the ideal effect of the vaccine. The good immune adjuvant not only can enhance the immunogenicity and the immune protective effect of the antigen, but also does not cause an allergic reaction or stimulate the self-immune reaction, and the like. The recombinant paramyosin molecule used in the study is expressed in E. coli, and the purity can reach more than 95% after the improved purification method, and is suitable for large-scale preparation and research purposes, the structure and biological characteristics of the recombinant protein and the worm-derived submuscle ball A combination of adjuvant and recombinant paramyosin was used to immunize mice, to find the best combination of the vaccine, and to explore the effect of the adjuvant of ISA206 and the recombinant paramyosin on the infection of Schistosoma japonicum. The ability to search for paramyosin phytophthora, which is suitable for the host of insect-protecting, such as cattle, and the like The combination of the seedlings. The present study was The results are as follows:1. The purity of the full-length paramyosin molecule expressed by the large-scale recombinant expression is over 95%, wherein the content of the LPS is 0.07 EU/ ml and the same secondary junction with the insect-derived paramyosin molecule Construction and biological functions, which include binding properties to immunoglobulins and with collagen the binding properties of the substance.2. In the Kunming mice and the C57BL/6 mouse model, rSj97-Hydrogel did not 3. Compared with rSj97-FA group and rSj97-CpG-ODN group, the rSj97-ISA206 group had significant protective effect on C57BL/6 mice compared with rSj97-FA group and rSj97-CpG-ODN group in the C57BL/6 mouse model. 4. Compared with the control group, the deinsectization rate was 16.08% (P = 0.742), the egg reduction rate of the liver was 3.64% (P = 0.963), and the egg reduction rate was 38. ..70% (P = 0.462). rSj97 induced a high level of specific IgG2a and IgG1 antibody in C57BL/6 mice, suggesting that rSj97 can induce the generation of specific Th1 and Th2-specific antibody subclasses, possibly related to the induction of C57BL/6 mice. 6. A high level of specific IgG2 and IgG1 antibodies can be induced by the rSj97 immune buffalo, and a Th1-type dominant response is induced, and humoral immunity and cellular immunity are induced. The response may be one of the factors that the buffalo produces a protective force. The study further enriched the study of the protective effect of paramyosin, especially in large animals, in order to build a suitable water buffalo
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392
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